Wulfert Sabine, Schilasky Sören, Krueger Stephan
Institute for Plant Sciences, University of Cologne, Zülpicherstraße 47b, 50674 Cologne, Germany.
Plants (Basel). 2020 Mar 11;9(3):353. doi: 10.3390/plants9030353.
Glycolysis is a central catabolic pathway in every living organism with an essential role in carbohydrate breakdown and ATP synthesis, thereby providing pyruvate to the tricarboxylic acid cycle (TCA cycle). The cytosolic pyruvate kinase (cPK) represents a key glycolytic enzyme by catalyzing phosphate transfer from phosphoenolpyruvate (PEP) to ADP for the synthesis of ATP. Besides its important functions in cellular energy homeostasis, the activity of cytosolic pyruvate kinase underlies tight regulation, for instance by allosteric effectors, that impact stability of its quaternary structure. We determined five cytosol-localized pyruvate kinases, out of the fourteen putative pyruvate kinase genes encoded by the genome, by investigation of phylogeny and localization of yellow fluorescent protein (YFP) fusion proteins. Analysis of promoter β-glucuronidase (GUS) reporter lines revealed an isoform-specific expression pattern for the five enzymes, subject to plant tissue and developmental stage. Investigation of the heterologously expressed and purified cytosolic pyruvate kinases revealed that these enzymes are differentially regulated by metabolites, such as citrate, fructose-1,6-bisphosphate (FBP) and ATP. In addition, measured in vitro enzyme activities suggest that pyruvate kinase subunit complexes consisting of cPK2/3 and cPK4/5 isoforms, respectively, bear regulatory properties. In summary, our study indicates that the five identified cytosolic pyruvate kinase isoforms adjust the carbohydrate flux through the glycolytic pathway in by distinct regulatory qualities, such as individual expression pattern as well as dissimilar responsiveness to allosteric effectors and enzyme subgroup association.
糖酵解是所有生物体内的一条核心分解代谢途径，在碳水化合物分解和ATP合成中起着至关重要的作用，从而为三羧酸循环（TCA循环）提供丙酮酸。胞质丙酮酸激酶（cPK）是一种关键的糖酵解酶，它催化磷酸烯醇丙酮酸（PEP）上的磷酸基团转移到ADP上以合成ATP。除了在细胞能量稳态中的重要功能外，胞质丙酮酸激酶的活性还受到严格调控，例如通过变构效应物，这些效应物会影响其四聚体结构的稳定性。通过对系统发育和黄色荧光蛋白（YFP）融合蛋白定位的研究，我们从基因组编码的14个假定丙酮酸激酶基因中确定了5个定位于胞质的丙酮酸激酶。对启动子β-葡萄糖醛酸酶（GUS）报告株系的分析揭示了这5种酶的亚型特异性表达模式，该模式因植物组织和发育阶段而异。对异源表达和纯化的胞质丙酮酸激酶的研究表明这些酶受到代谢物的差异调节，如柠檬酸、果糖-1,6-二磷酸（FBP）和ATP。此外，体外测定的酶活性表明，分别由cPK2/3和cPK4/5亚型组成的丙酮酸激酶亚基复合物具有调控特性。总之，我们的研究表明，所鉴定的5种胞质丙酮酸激酶亚型通过不同的调控特性，如个体表达模式、对变构效应物的不同反应以及酶亚组关联，来调节碳水化合物通过糖酵解途径的通量。
